Fertilized sea urchin eggs undergo a striking increase in oxygen consumption first described by Warburg over 80 years ago. The "respiratory burst" of fertilization is cyanide-insensitive and produces H202 as the substrate for an exocytosed peroxidase which crosslinks the protective protein coat surrounding the egg. Partially reduced oxygen species such as H202 damage cells, and these species are implicated in the pathogenesis of human disease. It is paradoxical that the sea urchin embryo generates large amounts of H202 at the beginning of development, suggesting that the production of this potentially lethal oxidant at fertilization is carefully controlled. We have isolated the membrane-associated oxidase responsible for H2O2 synthesis by the fertilized egg, and shown that it is specifically stimulated by protein kinase C. We now propose to extend our findings. First, we will explore the role of protein kinase C in activation of the respiratory burst by studying the purified kinase and by determining its primary structure. Second, we plan to identify the biochemical mechanism for H202 synthesis by the fertilized egg. These studies may reveal the electron transport chain protein(s) responsible for H202 generation, and should provide insights into regulation by protein phosphorylation. Third, we will determine if peroxidative mechanisms exist in mammalian fertilization, with implications for our understanding of early human development and contraception. Protein kinase C controls pathways critical for normal growth and differentiation, yet its downstream targets for regulation have proven difficult to identify. By studying the activation machinery for the respiratory burst oxidase, we hope to discover the molecular events that mediate signal transduction by this ubiquitous kinase.